A. Field of the Invention
The present invention relates to apparatus used to facilitate the laboratory production of small organic crystals in the approximate size range of 10 microns or less. More particularly, the invention relates to a micropositioner machine for extracting or “harvesting” individual micron-size protein crystals from a liquid in which the crystals are grown, and cryofreezing and storing the extracted crystals for subsequent crystallographic analysis.
B. Description of Background Art
The development of new therapeutic drugs by medical researchers, particularly those containing biochemicals, has in recent times benefited from an energizing technology in which individual protein crystals having particular structural characteristics are grown and tested for therapeutic efficiency. This technology is useful because of the discovery that interactions between proteins and other biochemicals with living organisms depend not only on the chemical composition of a biochemical, but also upon its physical structure. Thus, many biochemical reactions at the cellular level proceed at an accelerated rate if biochemical and cellular sites have complementary shapes, e.g., analogous to a triangular peg having an appropriate shape and size for fitting into a triangular recess, or vice versa. Conversely, a mismatch between the structures of a cellular binding site and a biochemical characterized by non-complementary shapes, results in situations somewhat analogous to trying to fit a square peg into a round hole. In such cases, the reaction rate between a cell and a biochemical may be unacceptably low.
For the foregoing reasons, medical and biochemical researchers have devoted increased attention to techniques for producing individual crystals, such as protein crystals, which have specific shapes or other structural characteristics.
According to one technique for producing protein crystals with particular from which a crystal is to be grown, along with some sort of agent such as a fragment of a crystal of the type to be grown, to act as a seed for initiating crystallization from the liquid, which is sometimes referred to as a liquor.
Typically, protein crystals having selected structures are developed by growing individual crystals from liquid contained in small individual wells formed in the upper surface of a plate. Typical plates used for the cultivation of protein crystals are rectangularly shaped, flat plates which are several centimeters on a side. Each plate has a matrix, typically rectangularly shaped, of separate, individual wells. For example, a 96-well “sitting-drop” plate may have 96 wells, each capable of holding a one-micro liter drop of crystal growth solution. Another typical cell cultivating plate has 24 2-micro liter wells.
After each well in a crystal growth plate has been filled with a desired volume of a crystal growth cocktail, a predetermined time period is allowed to elapse to thereby enable growth of crystals in each cell. The crystal growth plate is then positioned in the field of view of a stereo microscope, as a first step in extracting or “harvesting” individual crystals by a human or robotic operator.
According to a presently employed method of harvesting individual protein crystals from crystal growth wells, a human operator uses an elongated pick-up tool holder which has a small diameter planar pick-up loop protruding from its end. The pick-up or “harvester” loop sometimes referred to as a “cryoloop” is removably attached to the tool holder, has a diameter in the range of about 0.05 mm. to 1 mm., and is typically made of a looped filament of nylon, etched Kapton, or other hydrophobic material, which has a typical diameter of about 10 micrometers.
With the aid of the microscope, the human operator looks into the liquid cocktail to determine if one or more crystals are present. If more than one are present, they may be attached to each other and therefore need to be separated. The separation may be accomplished manually using a very small knife blade. Alternatively, the knife blade may be mounted on the tool arm of the micro manipulator machine for finer position control to separate the crystals.
With the aid of the microscope, the human operator inserts the harvester cryoloop into a solution in a well in which the crystals are grown, at an oblique angle to encircle a crystal contained in the solution. The tool, with the cryoloop and an attached liquid drop containing a crystal suspended in the solution by surface tension of the liquid, is then withdrawn upwardly from the crystal growth well.
A final step in harvesting individual crystals includes freezing a cryoloop holding a liquid drop and a crystal by exposing the loop and drop to a stream of a cryogenic gas, such as nitrogen evaporated from liquid nitrogen. This action, referred to as cryocooling or cryofreezing, freezes the crystals, liquid and cryoloop together, whereupon the cryoloop is removed from the tool holder, and placed in a cryogenic storage compartment cooled by a cryogenic fluid such as a liquid nitrogen or liquid propane.
Individual cryoloops each containing a crystal are subsequently analyzed by X-ray diffraction methods to determine whether the crystal has desired structural properties.
Because of the small sizes of protein crystals and the drops of liquid from which they form, it can be readily appreciated that the task of harvesting and storing the crystals requires moving the cryoloop in very small, precisely controllable increments. Accordingly, it would be desirable to provide an apparatus which has a capability for precisely manipulating a small cryoloop to extract small liquid drops containing protein crystals from growth wells, and cryofreezing the loop, liquid drop and crystals en masse for subsequent X-ray diffraction analysis and processing.
A method of Operator-Assisted Harvesting Of Protein Crystals Using A Universal Micro Manipulating Robot was described in an article of that title appearing in the Journal of Applied Crystallography (2007) 40. pp. 539-545. The entire contents of that article, which are directed to a fully automatic crystal harvesting method, are incorporated by reference into the present application.
The present invention was conceived of in part to provide a micro-manipulator machine for crystal harvesting which could substantially enhance the speed and precision with which a human operator could perform harvesting and preservation of protein crystals from small individual crystal growth wells.